NMR Study On The Structure And Function Of The Plant Protein AtGrp7 And The Oligosaccharide LNDFHI

In this thesis,liquid NMR is used as the main method to conduct a series of studies on the structures,interactions and functions of some proteins,ribonucleic acids and oligosaccharides that are closely related to the type III secretion system or gram-negative bacteria.?1?The expression and purification methods of AtGrp7 RRM domain from Arabidopsis thaliana were studied.The problem of impurities not being removed by regular purification was solved by denaturing-refolding protocol and acquired a sufficient amount of AtGrp7RRM domain.The structure of the AtGrp7 RRM domain was fully recovered by quick-dilution refolding,as supported by its finger-print ¹⁵N-¹H HSQC spectrum and CS-Rosetta model structures.ITC and NMR titration experiments further confirmed that the AtGrp7 RRM domain had the correct function with regards to RNA/DNA binding,which laied the foundation for further understanding the RNA/DNA chaperone function of this protein by the atomic level high-resolution NMR structure of the AtGrp7 RRM.?2?The backbone and side chain resonance assignment of the AtGrp7 RRM and the backbone resonance assignment of cyclophilin ROC1 were accomplished.On the basis of studying basic principle of protein NMR assignment and the specific procession was:peak picking,peak clustering,determine the peak which correspounds to the primary sequence of the protein and the side chain assignment,a preliminary study of the interaction between the AtGrp7 RRM and the effector protein HopU1 was completed.The binding site of HopU1with AtGrp7 was confirmed.?3?The problem of homologue-related TOCSY overlapped spectral peak in oligosaccharides was preliminarily solved by the modified 3D pulse sequence HSQC-TOCSY and the assignment was accelerated.The ¹H-¹H TOCSY spectrum of oligosaccharide was prepared using a 2 mmol/L lactose sample as a standard and the 3D NMR detection of low-concentration samples was effectively improved.The composition and structure of LNDFH I were determined by HSQC-TOCSY and regular series of 2D experiments.And the interaction between lectin LecB and LNDFH I was initially researched.